Since the red cell membrane is considered a model of many animal cell plasma membranes and because this model is used in the design of experiments relating to many tissues and diseases, it is important that the model be correct. The purpose of this proposal is to refine/correct certain aspects of the model that may be currently inaccurate, especially those aspects pertaining to the structure and function of the membrane's most abundant protein, band 3. First, the structure of the cytoplasmic domain of band 3 (cdb3) will be determined by x-ray crystallography, and the molecular basis of its functionally important conformational equilibrium will be evaluated by site-directed mutagenesis. Since cdb3 provides a membrane binding site for ankyrin, protein 4.1, protein 4.2, hemoglobin, hemichromes and several glycolytic enzymes, resolution of cdb3's 3-dimensional structure should allow refinement of the membrane's architecture at one of its major centers of organization. Second, the role of the band 3-ankyrin-spectrin bridge in determining membrane shape and mechanical properties will be evaluated. Because of the compelling nature of past research documenting the importance of the spectrin-based membrane skeleton in control of cell morphology/deformability, little attention has been directed towards the possible contribution of membrane-skeletal anchors in regulation of membrane properties. Therefore, methods that specifically sever the ankyrin tether to the membrane will be exploited to examine the importance of the protein bridge in maintaining membrane morphology/stability. Finally, the relationship between band 3 clustering and erythrocyte clearance will be quantitively measured in vivo, and the epitope of the naturally occurring a ti-band 3 IgG that promotes this clearance will be identified. Taken together, the proposed studies should expand our understanding of the molecular organization of the proteins that comprise a healthy erythrocyte membrane, and help clarify the changes that promote the rapid removal of an unhealthy/senescent cell from circulation.